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To generate the vaccine, 5.94 mL of virus stock ( 1*10^5 TCID50/mL)was treated with 60 mL of 3.7% formalin for 48 h at 37 °C. The inactivated virus (6 mL) was then dialyzed in 2 L of PBS at room temperature for 48h. Medium from uninfected Vero cells was treated in the same way to prepare mock vaccine.Vaccine was plated on Vero cells for 7 days to confirm inactivation. Vaccination was done intramuscularly at 0.5 mL per leg without adjuvant. The virus challenge ( 1x10^5 TCID50/mL) was split into 2 parts: 0.25 mL was given intratracheally, and 0.25 mL was given intranasally. A laryngoscope was used for the intratracheal portion, which was delivered via a 1-cc syringe with a 22-gauge plastic angiocatheter attached. The intranasal inoculum was delivered to each nostril via a plastic bulb–type pipettor.

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